Avidin, a glycoprotein found in chicken egg white as well as its distant relative, streptavidin from Streptomyces bacteria (Argarana, C. E. et al. Nucleic Acids Res 14, 1871-82 (1986)), have high affinity for biotin. This firm interaction has been utilized in countless applications in the different fields of life sciences to probe, label and affinity separate biomolecules. Another premise for the system has been the easy coupling of biotin to almost any other molecules without compromising the strong (strept)avidin-biotin bond or the function of the coupled molecule (Green, M. N. Advances in Protein Chemistry 29, 85-133 (1975), Wilchek, M. & Bayer, E. A. Biomol Eng 16, 1-4 (1999)).
Avidin is a homotetramer encoded by a single gene (Ahlroth, M. K. et al. Anim Genet 31, 367-75 (2000), Wales, M. J. et al. Gene 161, 205-9 (1995)). This fact together with the almost perfect 222 point structural symmetry (Pugliese, L. et al. Journal of Molecular Biology 231, 698-710 (1993)) and the orientation of the subunits in the protein guarantees that all four biotin-binding sites in avidin have equally high affinity towards biotin. From the evolutional point of view this also means that in the protein all four binding sites co-evolve. The monomers of avidin are simple classical up-and-down β-barrel proteins. They have identical topology of eight β-strands and their interconnecting loops (Pugliese, L. et al. Journal of Molecular Biology 231, 698-710 (1993), Hendrickson, W. A. et al. Proceedings of The National Academy of Sciences of the United States of America 86, 2190-2194 (1989), Livnah, O. et al. Proceedings of The National Academy of Sciences of the United States of America 90, 5076-5080 (1993)). If the 3-D structure of avidin is superimposed with that of streptavidin it is evident that the termini and also the biotin-binding pocket situates in topologically analogous regions in these proteins. Two monomer pairs in each avidin tetramer share a large common interface and they are therefore called as the structural dimers. The complete tetramer is composed of two of such structural dimers (Pugliese, L. et al. Journal of Molecular Biology 231, 698-710 (1993), Hendrickson, W. A. et al. Proceedings of The National Academy of Sciences of the United States of America 86, 2190-2194 (1989), Livnah, O. et al. Proceedings of The National Academy of Sciences of the United States of America 90, 5076-5080 (1993), Weber, P. C. et al. Science 243, 85-88 (1989)).
During the recent years avidin and streptavidin have been engineered via site-directed mutagenesis (Laitinen, O. H. et al. J Biol Chem 276, 8219-24 (2001), Laitinen, O. H. et al. FEBS Lett 461, 52-8 (1999), Marttila, A. T. et al. FEBS Lett 441, 313-7 (1998), Marttila, A. T. et al. FEBS Lett 467, 31-6 (2000), Reznik, G. O. et al. Proceedings of The National Academy of Sciences of the United States of America 95, 13525-30 (1998), Reznik, G. O. et al. Nat Biotechnol 14, 1007-11 (1996), Sano, T. et al. Proceedings of The National Academy of Sciences of the United States of America 94, 6153-6158 (1997), Sano, T. & Cantor, C. R. Proceedings of The National Academy of Sciences of the United States of America 92, 3180-3184 (1995), Chilkoti, A. et al. Bio/Technology 13, 1198-1204 (1995), Chilkoti, A. et al. Proceedings of The National Academy of Sciences of the United States of America 92, 1754-1758 (1995). Chu, V. et al. Protein Sci 7, 848-59 (1998), Freitag, S. et al. Proceedings of The National Academy of Sciences of the United States of America 96, 8384-9 (1999), McDevitt, T. C. et al. Biotechnol Prog 15, 391-6 (1999)). In some studies the focus has been on the adjustment of the physico-chemical properties of (strept)avidin whereas in other studies the target has been on the fine-tuning of the biotin-binding affinity. Nevertheless, as these mutant protein subunits are single gene products the desired changes, produced by mutations, take effect in all (strept)avidin subunits concurrently.
In several cases, however, it would be beneficial to alter for example the binding-affinity only in some subunits of the tetramer while conserving the tight binding in the rest of the binding sites. Chilkoti et al. have introduced a partial solution to this problem by producing separately two streptavidin forms, one having natural high-affinity biotin-binding capacity and another with reduced affinity (Chilkoti, A. et al. Bio/Technology 13, 1198-1204 (1995)). They denaturated these two forms and mixed them, after which the mixture was renatured. Nonetheless, the refolding led to many alternative forms: some of them contained four high affinity binding sites whereas other forms had a raising series of lower affinity binding sites, finally ending in the form that contained four lower affinity binding sites. In this sense the genetic fusion of the subunits might be more straightforward and effective strategy to create (strept)avidin molecules containing biotin-binding sites of variable affinity because it would lead into the uncoupling of the monomers as an evolutionary and protein engineering unit and therefore production of better quality preparations. The N- and C-termini of the distinct (strept)avidin subunits are, however, situated far away from each other in the quaternary structure and therefore it is presumable that any simple fusion strategy would fail.
A common approach to study protein folding and significance of secondary structure topology to the protein structure and function is the creation of circularly permuted forms of the examined proteins (Uliel et al. Protein Eng 14, 533-542 (2001)). Creation of circular permutations is an approach wherein the normal termini of a polypeptide are linked and new termini are created by breaking the backbone elsewhere. In many polypeptides, the normal termini are in close proximity and can be joined by a short amino acid sequence. The break in the polypeptide backbone can be at any point, preferably at a point where the natural function and folding of the polypeptide are not destroyed. In most cases proteins stand the circular permutations considerably well without exhibiting radical alterations in their structures or functions. Following circular permutation new C- and N-termini are created to the protein, allowing creation of fusion proteins wherein the fused peptide or protein is attached at a different place on the host protein.
Usually in this approach the original N- and C-termini are joined together with a linker peptide whereas the new termini are typically introduced into loop regions. Chu et al. have described a circularly permuted streptavidin that showed almost identical 3-D structure when compared to that of the native protein (Chu et al. Protein Sci 7, 848-59 (1998)). In that study the circular permutation strategy was used as a tool to delete the loop between α-strands three and four in streptavidin monomers. This loop is functionally important because it undergoes the open to closed conformational change upon biotin binding (Hendrickson et al. Proceedings of The National Academy of Sciences of the United States of America 86, 2190-2194 (1989), Weber et al. Science 243, 85-88 (1989)). Consequently, when the new termini were introduced in this loop, the affinity of the resultant mutant for biotin collapsed six orders of magnitude as compared to the wild-type (wt) streptavidin.
In U.S. Pat. No. 6,492,492 circularly permuted streptavidins were designed by altering one monomer but the monomers were not fused together. The resulting streptavidin has four monomers as the wt protein.